Project Summary Articular cartilage repair remains a challenge because of the lack of blood supply and accompanying posttraumatic inflammation. Despite the fact that autologous chondrocyte transplantation (ACT) is an option, cell source limitations retard the broad application of this approach clinically. Recently, there is increasing evidence indicating that adult stem cells are promising cell sources, particularly for stem cells derived from synovium (SDSCs), owing to its higher chondrogenic potential and lower hypertrophy. A small biopsy through arthroscopy can only provide a limited number of SDSCs for tissue regeneration, thus in vitro cell expansion is necessary; unfortunately, conventional expansion on plastic flasks causes cell senescence and loss of proliferation and differentiation capacity. Our recent reports indicated that decellularized extracellular matrix (dECM) deposited by SDSCs could enhance expanded stem cells' proliferation and chondrogenic potential. In this proposal, our central hypothesis is that dECM deposited by SDSCs can provide a superior tissue-specific matrix microenvironment for the optimal rejuvenation of human adult SDSCs in cartilage regeneration and defect repair. To achieve this hypothesis, we want to determine whether dECM deposited by SDSCs provides a superior matrix microenvironment for human adult SDSC rejuvenation compared to matrices from adipose- derived stem cells (ADSCs), urine-derived stem cells (UDSCs), or dermal fibroblasts (DFs) (Aim 1). We also plan to explore potential rejuvenation mechanisms by identifying specific matrix component(s) responsible for the rejuvenation of chondrogenic capacity via triggering the activation of critical integrin receptor(s) in expanded SDSCs (Aim 2). Lastly, a translational animal model will be used to evaluate SDSC/dECM repair strategies (Aim 3). Our objective is to determine the efficacy of this novel cell expansion system in providing a high quantity of high-quality SDSCs for the treatment of cartilage defects. This objective is consistent with our long-term goal which is to identify strategies for improved repair of cartilage defects in osteoarthritic patients using autologous stem cells. The primary impact of our expected findings would be significant not only in advancing the development of new generations of stem cell-based approaches for cartilage engineering and regeneration, but also in providing fundamental new knowledge regarding the interaction between stem cell and matrix microenvironment as well as potential mechanisms underlying stem cell rejuvenation by the surrounding stem cell matrix. Our dECM approach may also provide an excellent model for developing other tissue regeneration approaches.